Research Article

Received: 17 January 2011 Revised: 25 June 2011 Accepted: 29 June 2011 Published online in Wiley Online Library: 13 September 2011

(wileyonlinelibrary.com) DOI 10.1002/mrc.2817

Application of HR-MAS NMR spectroscopy for studying chemotype variations of Withania somnifera (L.) Dunal
S. K. Bharti,a Anil Bhatia,b S. K. Tewari,b O. P. Sidhub and Raja Roya
Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plants with a number of pharmaceutical and nutraceutical applications. Metabolic proling has been performed by HR-MAS NMR spectroscopy on fresh leaf and root tissue specimens from four chemotypes of W. somnifera. The HR-MAS NMR spectroscopy of lyophilized defatted leaf tissue specimens clearly distinguishes resonances of medicinally important secondary metabolites (withaferin A and withanone) and its distinctive quantitative variability among the chemotypes. A total of 41 metabolites were identied from both the leaf and root tissues of the chemotypes. The presence of methanol in leaf and root tissues of W. somnifera was detected by HR-MAS NMR spectroscopy. Multivariate principal component analysis (PCA) on HR-MAS 1 H NMR spectra of leaves revealed clear variations in primary metabolites among the chemotypes. The results of the present study demonstrated an efcient method, which can be utilized for metabolite proling of primary and secondary metabolites in medicinally important plants. Copyright c 2011 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article. Keywords: HR-MAS NMR; Withania somnifera; metabolic prole; principal component analysis

Introduction
Withania somnifera (L.) Dunal (Solanaceae) is one of the most important medicinal plants with a number of pharmaceutical and nutraceutical applications.[1] The extracts as well as different isolated bioactive constituents of W. somnifera have been reported to possess adaptogenic, anticancer, anti-convulsant, immunomodulatory, antioxidative and neurological effects.[2] The medicinal properties of W. somnifera have been attributed to the presence of unique classes of steroidal lactones called withanolides present in leaves and roots.[2] Several biologically active chemical constituents such as ashwagandhine, cuscohygrine, anahygrine, tropine, steroidal compounds, including ergostane-type steroidal lactones, withaferin A, withanolides, withasomniferin-A and withanone have been isolated from different parts of this plant.[3,4] Withaferin A and withanone are the major constituents of its leaves and roots which have been reported to show a potent angiogenesis inhibitor,[5] antitumor[6,7] and anticancer activity.[8,9] Earlier workers have reported a great phytochemical diversity with respect to withanolides content among different accessions of W. somnifera.[10,11] Recently, metabolite proling of W. somnifera extracts[12] and alterations in the levels of primary and secondary metabolites in its fruits at different developmental stages have been reported.[13] In plant systems, metabolomics relate changes in metabolite levels which are associated with yield, disease resistance, nutritional traits and gene function. In this context, metabolic proling has extensively been applied to a wide range of plants including peas,[14] tobacco,[15] tea,[16] grapes,[17] tomatoes,[18] wheat,[19] using various analytical methods. Among these, NMR spectroscopy is a commonly applied technique in plant metabolomics[20 24] and has been used to analyze a range of chemically diverse metabolites

including catechins, aliphatic compounds, aromatic compounds, organic acids, phenolics, fatty acids, steroids and sugars from extracts. NMR spectroscopy provides a rapid, non-destructive method which requires minimal sample preparation.[25] HR-MAS NMR spectroscopy is a further advancement of this technique to rapidly identify metabolites in tissues directly and to quantify those in order to follow metabolic shifts in plant metabolism. The analysis by HR-MAS NMR provides a complete characterization of the metabolites of the whole tissue without any extraction procedure. Its application has been recently reported in understanding the alterations in metabolite prole of Jatropha curcas due to Jatropha mosaic virus infection.[26] It has been proven that slight changes in the metabolome can be explained by perturbations imposed on plants. Perturbations include environmental change, physical stress, abiotic stress, nutritional stress, mutation and transgenic events. Variation of phytochemicals in different accessions of W. somnifera may constitute a serious obstacle for the quality control of herbal formulations that could lead to inconsistent medicinal properties. The present study supports the distinction amongst four

Correspondenceto:Raja Roy,CentreofBiomedical MagneticResonance,Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Raebareli Road, Lucknow 226 014, India. E-mail: rajaroy cdri@yahoo.com O. P. Sidhu, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India. E-mail: opsidhu@rediffmail.com

a Centre of Biomedical Magnetic Resonance, SGPGIMS Campus, Raebareli Road, Lucknow 226 014, UP, India b National Botanical Research Institute (Council of Scientic and Industrial Research), Rana Pratap Marg, Lucknow 226 001, UP, India

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S. K. Bharti et al. chemotypes, three of which have already been identied. In the present study, we have carried out HR-MAS NMR spectroscopy to evaluate variations in primary and secondary metabolites among different chemotypes of W. somnifera such as NMITLI-101 (rich in withaferin-A and withanone), NMITLI-108 (rich in withanone and lacks withaferin-A), NMITLI-118 (high withaferin-A with the absence of withanone)[10,27] and NBRI-WS. The objective of the present study was to investigate the range of metabolic variations in leaves and roots tissue of W. somnifera using HR-MAS NMR spectroscopy and to establish an efcient metabolic mapping approach for initial screening of primary and medicinally important secondary metabolites. All spectra were processed using a zero lling of 32 K and line broadening for exponential window function of 0.3 Hz prior to Fourier transformation. The HR-MAS 1 H NMR spectra of plant tissues were manually phased and automated baseline corrected using TOPSPIN 2.1 (Bruker Analytik, Rheinstetten, Germany). The 1 H NMR spectra were referenced to the methyl resonance of alanine at 1.48 ppm. The total analysis time (including sample preparation, optimization of NMR parameters and data acquisition) of 1 H HR-MAS NMR spectroscopy for each sample was approximately 30 min. The solution state 1 H NMR spectroscopic experiments from chloroform extracts of leaves of the chemotypes were performed on Bruker Biospin Avance 400 MHz FT NMR spectrometer in CDCl3 . To conrm the assignments, two-dimensional homo nuclear correlation spectroscopy (1 H 1 H COSY) and 1 H 13 C hetero nuclear single quantum correlation spectroscopy (HSQC) were performed using Brukers standard pulse program library. The parameters used for COSY were: 2048 data points were collected in the t2 domain over the spectral width of 12 820 Hz, 512 t1 increments were collected with 64 transients, relaxation delay of 1.5 s, acquisition time of 95 ms and presaturated water resonance during the relaxation delay. The resulting data were zero-lled to 1024 data points and were weighted with sine bell window functions in both the dimensions prior to Fourier transformation. The parameters used for 1 H 13 C HSQC were: 2048 data points were collected in t2 dimension over the spectral width of 12 820 Hz, 256 t1 increments were collected with 32 transients, relaxation delay of 2.0 s, acquisition time of 80 ms and a 90 pulse of 9.0 s. The phasesensitive data were obtained by the antiecho-time proportional phase increments (Antiecho-TPPI) method. The resulting data were zero-lled to 512 data points and were weighted with 90 shifted squared sine bell window functions in both the dimensions prior to Fourier transformation. Multivariate statistical analysis The CPMG spectra obtained from leaves (n = 32) and roots (n = 20) were individually subjected for further multivariate analysis of primary metabolites. The spectra were reduced to 951 (between 0.5 and 10.0 ppm) discrete chemical shift regions by digitization to produce a series of sequentially integrated regions 0.01 ppm width, using Bruker AMIX software (Version 3.8.7, Bruker GmbH). The region of 4.754.90 ppm was excluded from the analysis to remove the residual signal of HOD and water signal. The data obtained were mean centered, scaled to unit variance and then normalized by dividing each integral of the segment by the total area of the spectrum to compensate for the differences in the overall metabolite concentration between individual samples. The resulting data matrix was exported into Microsoft ofce Excel (Microsoft Corporation, USA). This was then further imported to The Unscrambler X Software package (Version 10.0.1, CamoUSA, Norway) for PCA analysis. The PCA analysis was also performed on the quantitated values of metabolites obtained from leaves and roots separately. The statistical signicance for the quantied metabolites was determined by one-way ANOVA post hoc Bonferroni multiple comparison test (SPSS 11.5.0, USA). A probability of p-value of <0.05 was taken to indicate statistical signicance.

Experimental
Sample collections and chemicals Four of chemotypes of W. somnifera: NMITLI-101, NMITLI-108, NMITLI-118 and NBRI-WS were investigated for variability in their metabolite proling using HR-MAS NMR spectroscopy. Root and leaf samples were collected from Experimental Research Station of the National Botanical Research Institute (NBRI), Lucknow, India. The three identied chemotypes are selections made from diverse habitats in different agro-climatic zones of India.[10,11] Their progenies are maintained at the Experimental Station of the institute. NBRI-WS is a new collection made by the authors. Fully developed leaf samples, sixth to seventh from the top; and samples from cortical region of root of nearly 150-day-old plants were collected during the rst week of March 2010. Collected samples were immediately frozen at 80 C until NMR analysis. A set of ve HR-MAS NMR measurements were performed for both the leaves and root tissue of all the samples. Deuterium oxide and CDCl3 used for NMR analysis were purchased from SigmaAldrich (St. Louis, MO, USA). Chemicals used for the spiking experiments were purchased from Merck Chemicals (Germany). NMR analysis
1 H NMR spectra were recorded on Bruker Biospin Avance-III 800 MHz NMR (Bruker GmBH, Germany) spectrometer operating at a proton frequency of 800.21 using 4-mm HR-MAS1 H/13 C/31 P triple resonance probe head equipped with magic angle gradient accessories. A zirconium oxide rotor of 4-mm diameter was used for the spectral recording with a spinning speed of 8000 1 Hz. Twenty microliters of deuterium oxide containing 0.03% (w/v) sodium salt of trimethylsilyl propionic acid (TSP; Sigma-Aldrich) was used for quantitative estimation of metabolites as well as for internal lock. Sample temperature was regulated using Bruker BCU-05 unit at 293 K during the acquisition of spectra. A sample (32 1.5 mg) of wet leaves and roots was used for the analysis. The 1 H NMR spectra with water suppression were acquired using onedimensional single pulse NOESY pulse sequence with the following experimental parameters: spectral width of 12820.5 Hz, mixing time of 100 ms (NOESY), time domain data points of 64K, effective 90 ip angle, 9.0 s, relaxation delay 4.0 s, acquisition time of 2.55 s, 64 number of scans with four dummy scans, a constant receiver gain of 50.8 with a total recording time of 9 min. The onedimensional CarrPurcellMeiboomGill (CPMG) pulse sequence with water suppression (echo time of 40 ms) and relaxation delay of 4.0 s was performed to remove short T2 components arising due to the presence of lipids as well as to obtain a good baseline for the quantitation as well as for multivariate analysis.

Results and Discussion

Identication of metabolites Metabolite variations among four chemotypes of W. somnifera were investigated using high-resolution magic angle spinning

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Fumaric aci d

Trigonelline

Trigonelline

UDP/Uridine

Phenylalanine

Formic acid

Adenosine

Tyrosine

Tyrosine

Trigonelline

Adenosine

Uracil

Uracil

UDP/Uridine

(B)

10.0

9.5

9.0
all sugars

8.5
Methanol

8.0

7.5

7.0

6.5

6.0

ppm

Myo-Inositol Choline Glucose

Malic acid / Citric acid

Succinic acid / Malic acid Glutamine Glutamic acid / Proline GABA Acetoacetate Glutamine Glutamic acid

Sucrose Fructose

GABA / Ornithine Unassigned Aspartic acid

Trigonelline Malic acid

Lactic acid/Threonine

Glucose

Glycine

GABA Lysine Arginine/Ornithine

Citric acid

Citrulline

Alanine

Valine

Isoleucine/Leucine

Galactose

(A) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Figure 1. A typical HR-MAS1 H NMR spectrum of Withania somnifera leaf showing different metabolites from expanded spectrum (A) from 0.5 to 4.7 ppm and (B) from 5.5 to 10.0 ppm.

(HR-MAS) NMR spectroscopy. Application of HR-MAS NMR spectroscopy supported distinction amongst the three chemotypes of W. somnifera already identied and conrmed the distinctness of the fourth accession to be regarded as a chemotype. NMR spectra of leaves and roots tissues were acquired in D2 O using the CPMG pulse sequence with water suppression to remove short T2 components arising due to the presence of lipids. The water suppression is required in the acquisition of NMR spectra to overcome the dynamic range problem and to recover metabolites that overlap with the broad baseline of the strong water resonance.[28] NMR spectra of all the chemotypes were acquired in D2 O and extensively analyzed by the combined use of one- and twodimensional NMR experiments. Highly resolved spectra were obtained and the assignment of the metabolites was further reinstated by comparing the 1 H spectra of reference compounds together with the existing literature values.[3,12,29,30] The reference compounds in Biological Magnetic Resonance Data Bank[31] were also used for the characterization of the metabolites. Recently, Perez et al. have reported similar resolution of 1 H HR-MAS NMR and solution 1 H NMR spectra of tomato fruit and tomato tissues with minimal sample manipulation for simultaneous analysis of polar and non-polar compounds.[32] The entire 1 H spectrum acquired in D2 O may be divided into three major regions. High-eld region (0.03.5 ppm) was rich with amino acids. Mid region (3.55.5 ppm)

contains sugars and down-eld (5.510.0 ppm) spectrum which was dominated by aromatic compounds. The 1 H NMR spectrum of leaf of W. somnifera chemotype NMITLI-101 acquired in D2 O using CPMG pulse sequence showed signals of acetoacetate, alanine, arginine, aspartic acid, choline, citric acid, citrulline, GABA, glutamine, glutamic acid, lactic acid, leucine, lysine, malic acid, methanol, myoinositol, ornithine, proline, succinic acid, threonine and valine in the high-eld region (Fig. 1). Signals from the mideld region showed diverse sugars included fructose, glucose (Glc), galactose and sucrose (Sucr) (Fig. 1). Signals from adenosine, formic acid, fumaric acid, phenylalnine, trigonelline, tyrosine, uracil, uridine and UDP were seen in the down-eld region (Fig. 1).The 1 H NMR spectrum of root tissue acquired in D2 O showed additional resonances of betaine and asparagine in the high-eld region (Supporting Information Fig. 1). The stack-plot of 1 H NMR spectra of all the chemotypes showed signal intensities of the identied metabolites in leaves (Fig. 2) and roots (Supporting Information Fig. 2). The 2D COSY spectra were extensively used to resolve the complexity of the overlapping/interfering spectral regions to identify the exact molecule in the tissue (Supporting Information Fig. 3). The fructose signals were assigned from the HSQC spectrum. Moreover, the singlet at 4.38 ppm was found to be the signal associated with trigonelline as its corresponding cross peak in the HSQC spectrum was observed at 50.9 ppm. The

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S. K. Bharti et al.
Alanine Lactate/Threonine

Trigonelline

Trigonelline

Methanol Myo-Inositol Glucose

GABA Unassigned Aspartic acid Malic acid Citrate

Trigonelline Malic acid Sucrose Fructose

Trigonelline

Glucose

Sucrose

Glucose

GABA

6X 6X 4X

(D)

(C)

(B)

(A) 8.0 8.0 7.0 5.4 5.2 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Figure 2. Stack-plot of HR-MAS 1 H NMR single pulse spectra of leaves of Withania somnifera (A) NMITLI-101, (B) NMITLI-108, (C) NMITLI-118 and (D) NBRI-WS. All the four spectra were plotted on the same intensity scale.

(h)

(g)

(f)

(e)

(d)

1 H NMR spectra of the roots were found to be mainly dominated by the presence of sucrose. A HR-MAS NMR spectroscopy protocol was standardized to investigate qualitative and quantitative variability in withaferin A and withanone from leaf tissue of W. somnifera. The proton HRMAS NMR spectra of defatted lyophilized leaves were acquired in CDCl3 using CPMG pulse sequence. Comparison of the spectrum with the puried withanolide standards established the presence of withaferin-A and withanone in the leaves of W. somnifera. The 1 H NMR spectra revealed the presence of withaferin-A and withanone in leaves. The HR-MAS NMR spectra were similar in resolution when compared with solution state NMR of leaf extracts in all the four chemotypes (Fig. 3) with the advantage of minimal sample preparation. A total of 41 metabolites were identied from both the leaves and roots of all the four chemotypes (Table 1).

Quantitation of metabolites
(c)

(b)

(a)

7.0

6.8

6.6

6.4

6.2

6.0

ppm

Figure 3. Comparison of solution-state NMR spectra of leaf extracts of Withania somnifera in CDCl3 and its HR-MAS 1 H NMR spectra of defatted lyophilized leaves (a and b) NMITLI 101, (c and d) NMITLI 108, (e and f) NMITLI 118 and (g and h) NBRI-WS.

NMR spectral complexity (overlapping signals) did not allow quantication of all the metabolites. However, 16 of them were quantied by integrating the distinct characteristic signals of each metabolite with respect to the intensity of the nine protons of TSP (in D2 O, 0.375%, w/v) on the fresh weight basis, and in CDCl3 on dry weight basis.[26,33] The quantitative analysis was carried out on CPMG NMR spectra as it provided proper baseline. Earlier workers[34,35] have used T1 correction factor (Eqn 1) for the quantication of metabolites using single pulse NMR spectra. The application of the T1 correction factor may not be appropriate for the quantication in the CPMG NMR spectra, as shorter relaxation delays introduces inaccuracy in the measured concentration due to combined effect of T1 and T2 relaxation. Therefore, for absolute quantication, T1 and T2 of metabolites in the wet leaves and root tissue specimens using

1H

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Table 1.

H chemical shift assignments of the metabolites of different chemotypes of W. somnifera Plant parts

S.N. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Metabolites Alanine Acetic acid Acetoacetate Adenosine Aspartic acid Arginine Asparagine Betaine Choline Citric acid Citrulline Fructose Fumaric acid Formic acid GABA Galactose Glutamic acid Glutamine Glucose Glycine Isoleucine Lactate Leucine Lysine Malic acid Methanol Myoinositol Ornithine Phenylalanine Proline Succinic acid Sucrose Tyrosine Threonine Trigonelline Uracil Uridine UDP Valine Withaferin A Withanone

Chemical shift (multiplicity) 1.48 (d), 3.78 (q) 1.92 (s) 2.23 (s) 8.21 (s), 8.26 (s), 6.02 (d) 2.652.78 (m), 3.90 (dd) 1.70 (m), 1.91 (m), 3.24 (t), 3.76 (dd) 2.852.91 (m), 2.91 (dd) 3.28 (s) 3.21 (s) 2.672.80 (m) 1.56 (m), 1.87 (m), 3.13 (t) 4.10 (d), 4.03 (d), 4.02 (d) 6.52 (s) 8.45 (s) 1.94 (m), 2.31 (t), 3.02 (t) 4.58 (d), 5.26 (d), 3.49 (t) 2.07 (m), 2.35 (t), 3.74 (m) 2.13 (m), 2.44 (m), 2.73 (m) 5.25 (d), 4.65 (m), 3.25 (t) 3.57 (s) 0.92 (t), 0.99 (d) 1.33 (d), 4.11 (q) 1.72 (m), 0.95 (dd) 1.88 (m), 1.72 (m), 1.50 (m) 4.35 (m), 2.492.73 (m) 3.36 (s) 4.04 (t), 3.61 (t), 3.51 (dd), 3.28 (t) 3.02 (t), 1.77 (m) 7.357.43 (m) 2.35 (m), 2.08 (m), 2.01 (m) 2.38 (s) 5.42 (d), 4.22 (d), 4.04 (t), 3.68 (s) 6.88 (d), 7.17 (d) 1.34 (d), 4.22 (m) 9.13 (s), 8.83 (d), 7.87 (t), 4.44 (s) 5.8 (d), 7.57 (d) 5.97 (m), 7.98 (d) 5.98 (d), 7.96 (dd) 1.04 (d), 1.01 (d) 6.95 (dd), 6.21 (d) 6.60 (dd), 5.85 (d)

Leaves a,b B B B a,b B b ND a,b b b b b b a,b b b b a,b b b a,b b b a,b a,b a,b b b b b a,b b b a,b b b b b a,b a,b

Root a,b b b ND b b a,b b a,b b ND ND b b a,b ND ND B A,b B B a,b b b a,b a,b b b b b b a,b b b ND b ND ND B

a, Quantied metabolites; b, detected only; ND, not detected.

HR-MAS NMR spectroscopy were calculated by the application of classical inversion recovery method and CPMG pulse sequence. The respective T1 and T2 correction factors for each metabolite were simultaneously applied for the quantication in the CPMG spectra using the following equation 2, as per our earlier work:[36] Conc() = Conc(t) (1 e (1 e
t/T1 S

M denote reference and metabolites, respectively Conc(a) = Conc(cpmg) expts /T2 expts /T2
S

1 expt/T1 1 expt/T1

(2)

) )

t /T 1 M

(1)

where Conc() and Conc(t) are the concentration calculated at full relaxation and incomplete relaxation time t, respectively. S and

where Conc(a) is the absolute concentration of metabolites, Conc(cpmg) is the concentration of metabolites calculated by CPMG pulse sequence at shorter repetition time and a xed echo time. Tissue-specic metabolites were observed in W. somnifera. Quantitative variability of metabolites in both the leaves and roots of all chemotypes is shown in Fig. 4. Myoinositol and aspartic acid were observed only in leaf tissues of all the four chemotypes.

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0.10 0.08 Conc. (mg/g) 0.06 0.04 0.02 0.00 A 0.03 Conc. (mg/g) B

Alanine. 0.5 0.4 0.3 0.2 0.1 0.0 C Betaine. Conc. (mg/g) D 0.4 0.3 0.2 0.1 0.0 A B C Glucose. Conc. (mg/g) D 0.8 0.6 0.4 0.2 0.0 A B C D 0.8 Conc. (mg/g) 0.6 0.4 0.2 0.0 A B C Sucrose. Conc. (mg/g) D 0.5 0.4 A A A A

Aspartic Acid. Conc. (mg/g)

2.5 2.0

Asparagine.

Conc. (mg/g)

1.5 1.0 0.5 0.0

C Choline

C GABA.

0.25 Conc. (mg/g) 0.20 0.15 0.10 0.05 0.00

0.02

0.01

0.00

D 0.8 Conc. (mg/g) 0.6 0.4 0.2 0.0

C Lactic Acid.

5 4 Conc. (mg/g) 3 2 1 0

Glutamine.

B Methanol.

D 6 Conc. (mg/g)

3 Conc. (mg/g)

Malic Acid.

Myo-Inositol.

0 B C D 15 Conc. (mg/g) A B Withanone C D

60 Conc. (mg/g)

Trigonelline.

40

10

0.3 0.2 0.1

20

0 A 25 20 Conc. (mg/g) 15 10 5 0 A B C D B C D

0.0 A B C D

0 A B C D

Withaferin-A Leaf Root A: NMITLI 101 B: NMITLI 108 C: NMITLI 118 D: NBRI-WS

Figure 4. Quantitative variability in polar and semi-polar metabolites among different chemotypes of Withania somnifera. Error bars = SD.

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Application of HR-MAS NMR for metabolic proling of Withania somnifera

(B)
Glucose Glucose Glucose

0.2

Choline Methanol

Unassigned

0.1 NMITLI 118 NMITLI 101 NBRI-WS 0

Malic acid

-0.2

PC-2 (23%)

5.83

5.27

4.56

3.99

Sucrose

Myoinositol

3.42

Myoinositol

0.05

-0.1
Sucrose Sucrose

2.85

2.28

1.72

1.15

Unassigned

(A)

NMITLI 108

Alanine Lactic acid

0.58

X-variables (PC-1) (56%)

Myoinositol Trigonelline Methanol Myoinositol Choline

(C) -0.05 -0.1 -0.05 0 PC-1 (56%) 0.05 0.1

0.1 0.2

0
Malic acid GABA Glucose Glucose GABA GABA

Unassigned

-0.1
Sucrose

6.08

5.49

4.75

4.15

3.56

2.96

2.37

1.77

Alanine

Lactic acid

1.17

0.58

X-variables (PC-2) (23%)

Figure 5. (A) PCA scores plot obtained from the PC analysis of HR-MAS1 H NMR spectra of Withania somnifera leaves. PC1 and PC2 explained 79% of the total variance. Loadings plot for principal component 1 (B) and principal component 2 (C).

The concentration of myoinositol ranged from 1.7 0.18 to 5.3 0.26 mg/g fresh weight leaf, the lowest being in NMITLI-108 and highest in NMITLI-118. The myoinositol has been reported to be directly associated with osmoprotectant[37] properties of plants. Asparagine, betaine and glutamine were found to be present only in root tissue of NMITLI-101, NMITLI-108 and NMITLI118. The concentration of asparagine ranged from 0.65 0.11 to 1.58 0.32 mg/g fresh weight root, the lowest being in NMITLI101 and highest in NMITLI-118. Asparagine was not detected in case of NBRI-WS. Glutamine content was nearly equal in NMITLI101 and NMITLI-118 (0.50 0.04 and 0.43 0.05 mg/g fresh weight root) and was found to be approximately 32% higher in NMITLI-108. The concentration of betaine was nearly equal in NMITLI-101 and NMITLI-108; however, it was not detected in other two chemotypes. Betaine content has been reported to increase several-fold under salt stress conditions.[38] The glutamine and asparagine serve as the major nitrogen transport compounds in higher plants.[39] Alanine, choline, GABA, glucose, lactic acid, malic acid, methanol, sucrose and trigonelline were observed in both the root and leaf tissues. The concentration of these metabolites was varied signicantly in leaves and roots of different chemotypes. Choline content was higher in leaves as compared with that of root tissue, whereas lactic acid showed a trend

reverse to that of choline. Alanine content was nearly equal in leaves and roots of NMITLI-101, NMITLI-108 and NBRI-WS; however, it was more than twofold higher in root as compared with that of leaves in NMITLI-118. The concentration of glucose was higher in leaves as compared with that of root in all the chemotypes of W. somnifera. However, sucrose showed a trend reverse to that of glucose in all the chemotypes. The concentrations of withaferin-A and withanone varied signicantly among different chemotypes (Fig. 4). Resonances of withaferinA were clearly resolved in NBRI-WS and NMITLI-108, whereas well-resolved signals of withanone were observed in NMITLI101, respectively (Fig. 4). In the present study, a small change in the quantitative value of withaferin-A and withanone values was observed in NMITLI-101, 108 and 118. This may be due to the multiplication of the chemotypes in the eld through reproduction using seeds. Literature survey revealed that methanol in the plant leaf and root tissues has not been reported. However, recently the presence of methanol in the 1 H NMR spectrum of buffer extract without pre and post-lyophilization treatment has been observed in fresh tobacco.[40] The extraction procedures usually tend to lose the NMR signal of methanol due to its low boiling point. The singlet at 3.36 ppm in 1 H NMR spectrum indicated

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Table 2. The statistical signicance for various metabolites was determined by one-way ANOVA with post hoc Bonferroni multiple comparisons test Metabolites Alanine Asparagine Aspartic Acid Betaine Choline GABA Glutamine Glucose Lactic acid Malic acid Methanol Myoinositol Sucrose Trigonelline Plant part L R L R L R L R L R L R L R L R L R L R L R L R L R L R A vs. B NS NS ND NS

A vs. C

A vs. D NS

B vs. C NS NS ND NS

B vs. D NS

C vs. D NS NS ND

NS ND

ND ND NS NS

NS ND ND

ND NS NS ND ND

ND

ND ND

ND ND

NS

NS

NS

NS NS

NS ND ND ND NS

NS

ND NS

NS NS NS

NS ND NS NS NS

NS ND

NS ND

ND

NS ND

NS NS

NS

NS NS NS

NS NS

NS NS NS

NS NS NS

ND

NS NS NS NS ND

NS

NS NS NS

NS

NS

ND

NS NS ND

NS

ND NS NS

ND NS

ND

ND

ND

ND

ND

ND

NS, not signicant; ND, not detected; L, leaves; R, roots; A, NMITLI-101; B, NMITLI-108; C, NMITLI-118; D, NBRI-WS. Number of subjects per group was eight for leaves and ve for roots. Signicant at p 0.05; p 0.01.

the presence of methanol. The 1 H NMR spectra of lyophilized leaves showed the absence of the signal at 3.36 ppm and spiking experiment using pure methanol in fresh leaf tissues clearly indicated the presence of methanol in W. somnifera. The present study could also quantify the methanol in the plant tissues. In order to substantiate our nding three plant species from Solanaceae family such as Capsicum annum L. (Chilli), Solanum lycopersicum L. (tomato) and Datura stramonium L. were subjected to HR-MAS NMR analysis (Supporting Information Fig. 4). The concentration of methanol was higher in leaves than that of root in all the four chemotypes (Fig. 4). The emission of methanol by plants was rst demonstrated by converting it into furfural followed by GC analysis.[41] Its emission from vegetation is quantitatively linked to the process of pectin demethylation.[42] Principal component analysis of 1 H HR-MAS NMR spectra of leaves Multivariate principal component analysis (PCA) was used on the 1 H NMR spectra of leaves of all the chemotypes. A sevencomponent model explained 97.0% of the variance, with the rst two components explaining 79.0% of the total variance. The clear clustering between all the chemotypes in the PCA of leaves spectra demonstrated signicant variations of metabolites (Fig. 5). Examination of PC1 loadings showed that the cluster separation

arises due to positive loadings of glucose, choline, methanol, alanine and threonine/lactate and negative loadings of sucrose, myoinositol and malic acid. This suggested that metabolites from NMITLI-118 and NMITLI-101 had relatively lower concentration of glucose, choline, methanol, alanine and threonine/lactate and relatively higher concentrations of sucrose, myoinositol and malic acid than other chemotypes. Some intra-variability within each groups were also observed. The examination of PC2 loadings showed that the cluster separation was due to the positive loadings of trigonelline, myoinositol, methanol, choline and threonine/lactate and negative loadings of sucrose, glucose, GABA, malic acid and alanine. This suggested that samples from NMITLI-101 and 108 had relatively lower concentration of trigonelline, myoinositol, methanol, choline, threonine/lactate and relatively higher concentrations of sucrose, glucose, GABA, malic acid and alanine. These also suggested that NMITLI-118 had relatively higher concentrations of trigonelline, myoinositol, methanol, choline and threonine/lactate and relatively lower concentrations of sucrose, glucose, GABA, malic acid and alanine than that of NBRI-WS. The regions from 5.5 to 10.0 ppm (except trigonelline) were not playing any signicant role in the PCA of leaves. The PCA was also applied on the 1 H HR-MAS NMR spectra of roots of all the chemotypes. A seven-component model explained 96.40% of the variance, with the rst two components explaining 74.0% of the total variance. The score plot (Supporting Information Fig. 5) did not show clear separation

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Application of HR-MAS NMR for metabolic proling of Withania somnifera between chemotypes. The 16 quantied metabolites from leaves and roots of all the chemotypes were subjected to univariate statistical analysis. The statistical signicance of those metabolites is reported in Table 2. The multivariate PCA was also performed on the quantied metabolites from leaves and roots (Supporting Information Fig. 6) and produced similar results when compared with the PCA of leaves and roots spectra. The univariate result also correlates well with the PCA on the quantied data, respectively.
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Conclusion
HR-MAS NMR spectroscopy based metabolite proling approach covers a large variety of metabolites, mainly those involved in primary metabolism, including amino acids, sugars, sugar alcohols and other intermediates in the fresh leaf and root tissues. For biologically active secondary metabolites such as withanolides (withaferin A and withanone), a separate HR-MAS experiments were conducted on defatted lyophilized leaf tissues. HR-MAS NMR spectroscopy was found to be a quick and an efcient technique to discriminate different chemotypes of W. somnifera. PCA, performed on the 1 H HR-MAS NMR spectra, revealed a clear separation among chemotypes even with respect to primary metabolites. Metabolomic ngerprinting of herbal extracts is desirable to standardize drugs and to establish the scientic basis of their pharmacological action. This study recruited HR-MAS NMR technique for rapid metabolomic analysis of leaf and root tissues of different chemotypes of W. somnifera. Such knowledge will evolve directions for genetic improvement of medicinal plants for the enhancement of pathways leading to the biosynthesis of bioactive molecules. The study supports the distinction of chemotypes in W. somnifera. Acknowledgements The nancial support from the Council of Scientic and Industrial Research, New Delhi, under a NMITLI (New Millennium Indian Technology Leadership Initiative) program is gratefully acknowledged. The authors are thankful to Dr. C. S. Nautiyal, Director NBRI, Lucknow and Professor C. L. Khetrapal, Director C.B.M.R., (S.G.P.G.I.M.S. campus), Lucknow, for providing facilities. Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, is gratefully acknowledged for developing the chemotypes of W. somnifera. Supporting information Supporting information may be found in the online version of this article.

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